Screening method for pain suppressor and pharmaceutical composition for prevention or treatment of pain

ABSTRACT

The present invention provides a screening method for a pain suppressor, which method comprises using FLRT3 to select a substance capable of inhibiting FLRT3 expression or a substance capable of inhibiting FLRT3 transport to the spinal cord. In addition, the present invention provides a pharmaceutical composition for prevention or treatment of pain, which pharmaceutical composition comprises, as an active ingredient, a substance capable of inhibiting FLRT3 expression or a substance capable of inhibiting FLRT3 transport to the spinal cord.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCTInternational Application Number PCT/JP2016/053919, filed on Feb. 10,2016, designating the United States of America and published in theJapanese language, which is an International Application of and claimsthe benefit of priority to Japanese Patent Application No. 2015-025954,filed on Feb. 13, 2015. The disclosures of the above-referencedapplications are hereby expressly incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isSeqList-IWAT007-001APC.txt, the date of creation of the ASCII text fileis May 30, 2017, and the size of the ASCII text file is 4 KB.

TECHNICAL FIELD

The present invention relates to a screening method for a painsuppressor. The present invention also relates to a pharmaceuticalcomposition for treatment or prevention of pain.

BACKGROUND ART

Pain is an intractable disease which reduces the QOL (Quality of Life)of the affected individuals. The number of chronic pain sufferers in theworld is reported to exceed 20 million, and the combined market size ofJapan, the U.S. and Europe for pain medicines is said to be about 2trillion yen. In addition, the number of patients with diseases whichmay be a cause of pain, such as stroke, cancer, diabetes and viralinfection, has been increasing, and under such circumstances, theestablishment of an appropriate treatment modality for pain is a veryimportant medical issue. Particularly, neuropathic pain is lesssensitive to nonsteroidal antiinflammatory drugs and opioid analgesics,and is a disease with significant unmet medical needs. However, thepathogenesis of neuropathic pain is diverse and the underlying molecularmechanism is very complicated. Therefore, radical therapeutic medicinesfor neuropathic pain are yet to be developed (Non Patent Literature 1).Clarifying the molecular mechanism of the development and maintenance ofneuropathic pain leads to the development of breakthrough medicines andis expected to provide a promising solution to one of the biggestmedical issues in the 21st century.

The dorsal horn of the spinal cord is considered to be one of the majorcausative site of neuropathic pain (Non Patent Literature 2). Peripheralsensory inputs undergo various processing, such as amplification,attenuation and integration, in the dorsal horn of the spinal cord, andare transmitted to the brain. However, injury of peripheral nervesreportedly induces neural network changes in the dorsal horn of thespinal cord, for example abnormal axon collateral formation and enhancedsynaptic transmission, thus triggering pain (Non Patent Literature 3).Therefore, clarifying a novel molecular mechanism which regulates theneural circuit in the dorsal horn of the spinal cord is expected to leadto the discovery of a novel target molecule required for the developmentof breakthrough medicines for pain.

FLRT3 (fibronectin and leucine-rich transmembrane protein-3) is a memberof the fibronectin leucine-rich repeat transmembrane protein family andhas a FN (fibronectin) type III domain and leucine-rich repeats. FLRT3is expressed in various tissues including kidney, skeletal muscle, brainand lung (Non Patent Literature 4). FLRT3 is reported to play importantroles in the cell and tissue morphogenesis during early embryogenesis,including neurite formation (Non Patent Literature 5), cell adhesion(Non Patent Literature 6), axon guidance (Non Patent Literature 7), etc.Also reported is that, after peripheral nerve injury, FLRT3 proteinexpression increases at the axon terminals of dorsal root ganglionneurons projecting into the dorsal horn of the spinal cord (Non PatentLiterature 8). Moreover, FLRT3 is reported to bind to the Unc5B receptor(Non Patent Literature 7 and 9). However, there have been no studyreports on the in vivo functions of FLRT3 in the adult spinal cord, andit has yet to be clarified whether FLRT3 is involved in the developmentof neuropathic pain.

CITATION LIST Non Patent Literature

-   Non Patent Literature 1:-   Dworkin, R. H. et al. Pharmacologic management of neuropathic pain:    evidence-based recommendations. Pain 132, 237-51 (2007).-   Non Patent Literature 2:-   Woolf, C. J. Neuronal Plasticity: Increasing the Gain in Pain.    Science 288, 1765-1768 (2000).-   Non Patent Literature 3:-   Markman J. D. & Dworkin, R. H. Ion channel targets and treatment    efficacy in neuropathic pain. The journal of pain 7, S38-47 (2006).-   Non Patent Literature 4:-   Lacy, S. E., Bo, C. G., Buzney, E. A., Kunkel, L. M. & Al, L. E. T.    Identification of FLRT1, FLRT2, and FLRT3: A Novel Family of    Transmembrane Leucine-Rich Repeat Proteins. 426, 417-426 (1999).-   Non Patent Literature 5:-   Tsuji, L. et al. FLRT3, a cell surface molecule containing LRR    repeats and a FNIII domain, promotes neurite outgrowth. Biochemical    and Biophysical Research Communications 313, 1086-1091 (2004).-   Non Patent Literature 6:-   Chen, X., Koh, E., Yoder, M. & Gumbiner, B. M. A    protocadherin-cadherin-FLRT3 complex controls cell adhesion and    morphogenesis. PloS one 4, e8411 (2009).-   Non Patent Literature 7:-   Yamagishi, S. et al. FLRT2 and FLRT3 act as repulsive guidance cues    for Unc5-positive neurons. 1-14 (2011). doi: 10.1038/emboj.2011.189-   Non Patent Literature 8:-   Robinson, M. et al. FLRT3 is expressed in sensory neurons after    peripheral nerve injury and regulates neurite outgrowth. Molecular    and cellular neurosciences 27, 202-14 (2004).-   Non Patent Literature 9:-   Seiradake E. Et al. FLRT structure: balancing repulsion and cell    adhesion in cortical and vascular development. Neuron, 84(2),    370-385 (2014).

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to find a molecule involved inperipheral neuropathic-associated pain and to provide a screening methodfor a pain suppressor. Another object of the present invention is toprovide a pharmaceutical composition for prevention or treatment ofpain, which composition comprises a novel active ingredient.

Solution to Problem

-   The present invention includes the following to achieve the    above-mentioned objects.-   (1) A screening method for a pain suppressor, the method comprising    using FLRT3.-   (2) The method according to the above (1), wherein the pain    suppressor is a substance capable of suppressing peripheral    neuropathic-associated pain.-   (3) The method according to the above (1) or (2), wherein a    substance capable of inhibiting FLRT3 expression is selected.-   (4) The method according to the above (1) or (2), wherein a    substance capable of inhibiting FLRT3 transport to spinal cord is    selected.-   (5) The method according to the above (1) or (2), wherein a    substance capable of inhibiting FLRT3 binding to Unc5B is selected.-   (6) The method according to the above (3), comprising the steps of:

bringing a test substance into contact with cells expressing FLRT3;

measuring the level of FLRT3 expression in the cells; and

comparing the level of FLRT3 expression to that in FLRT3-expressingcells not in contact with the test substance to determine whether or notthe test substance is capable of reducing the level of FLRT3 expression.

-   (7) The method according to the above (4), comprising the steps of:

bringing a test substance into contact with dorsal root ganglion cellsin dorsal root ganglion tissue;

measuring the level of FLRT3 in dorsal root fibers or spinal cord; and

comparing the level of FLRT3 to that in dorsal root fibers or spinalcord in the absence of contact with the test substance to determinewhether or not the test substance is capable of reducing the level ofFLRT3.

-   (8) The method according to the above (5), comprising the steps of:

bringing a test substance into contact with FLRT3 and Unc5B;

assessing FLRT3 binding to Unc5B; and

determining whether or not the test substance is capable of inhibitingFLRT3 binding to Unc5B.

-   (9) A pharmaceutical composition for prevention or treatment of    pain, comprising, as an active ingredient, a substance capable of    inhibiting FLRT3 expression, a substance capable of inhibiting FLRT3    transport to spinal cord, or a substance capable of inhibiting FLRT3    binding to Unc5B.-   (10) The pharmaceutical composition according to the above (9),    wherein the pain is peripheral neuropathy-associated pain.-   (11) The pharmaceutical composition according to the above (9) or    (10), wherein the substance capable of inhibiting FLRT3 expression    is a nucleic acid capable of inhibiting FLRT3 expression.-   (12) The pharmaceutical composition according to the above (9) or    (10), wherein the substance capable of inhibiting FLRT3 transport to    spinal cord or the substance capable of inhibiting FLRT3 binding to    Unc5B is an antibody or a peptide capable of specifically binding to    FLRT3.-   (13) The pharmaceutical composition according to anyone of the    above (9) to (12), wherein the pharmaceutical composition has an    action at dorsal root ganglion cells, dorsal root fibers or axon    terminals of dorsal root ganglion neurons.

Advantageous Effects of Invention

The screening method of the present invention enables the discovery of apain suppressor useful as a preventive or therapeutic medicine for pain.In addition, the pharmaceutical composition of the present invention isuseful for prevention or treatment of pain.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of quantitative PCR analysis of the FLRT3 geneexpression level in dorsal root ganglion cells harvested fromneuropathic pain model rats.

FIG. 2 shows the results of quantitative PCR analysis of the FLRT3 geneexpression level in the spinal cord harvested from neuropathic painmodel rats.

FIG. 3 shows the results of western blotting analysis of the FLRT3protein expression level in dorsal root ganglion cells harvested fromneuropathic pain model rats.

FIG. 4 shows the results of western blotting analysis of the FLRT3protein expression level in the spinal cord harvested from neuropathicpain model rats.

FIG. 5 shows the change in withdrawal threshold in rats in the von Freyfilament test after intrathecal administration of FLRT3 protein.

FIG. 6 shows the time-course change in withdrawal threshold in the hindlimb ipsilateral to injury in neuropathic pain model rats in the vonFrey filament test after intrathecal administration of anti-FLRT3antibody.

DESCRIPTION OF EMBODIMENTS

Screening Method

The present invention provides a screening method for a pain suppressor.The screening method of the present invention comprises using FLRT3. TheFLRT3 used in the screening method of the present invention may be aprotein or a gene. In the case where the FLRT3 is a protein, the proteinmay be a full-length one or a functional fragment thereof.

The FLRT3 used in the screening method of the present invention may beof any living organism and is not particularly limited. Preferred is amammalian FLRT3. The mammal is preferably a human, a chimpanzee, amonkey, a dog, a cow, a mouse, a rat, a guinea pig or the like, and ismore preferably a human. Information regarding the nucleotide sequencesof the genes encoding FLRT3 proteins of various animals and the aminoacid sequences of the FLRT3 proteins can be obtained from knowndatabases (e.g., DDBJ/GenBank/EMBL) with the respective accessionnumbers shown in Table 1, for example.

TABLE 1 Nucleotide sequence Amino acid sequence Human AY358319 AAQ88685Rat NM_001126291 AAI60843 Mouse NM_001172160 AAH52043

The test substances to be screened are preferably nucleic acids,peptides, proteins, non-peptidic compounds, synthetic compounds,fermentation products, cell extracts, cell culture supernatants, plantextracts, mammalian tissue extracts and plasma, etc., but are notlimited to these examples. The test substances may be novel or knownsubstances. These test substances may be in the form of a salt. The saltis composed of the test substance with a physiologically acceptable acidor base.

In the screening method of the present invention, it is preferred toselect a substance capable of inhibiting FLRT3 expression or a substancecapable of inhibiting FLRT3 transport to the spinal cord. The presentinventors have found that a rat partial sciatic nerve ligation modelshows a significant increase in FLRT3 gene and protein expression indorsal root ganglion cells on the side ipsilateral to the injury,followed by the transport of the expressed FLRT3 protein to the spinalcord through dorsal root fibers (axons), resulting in pain amplification(see Examples). Therefore, a substance capable of inhibiting FLRT3expression in dorsal root ganglion cells and a substance capable ofinhibiting FLRT3 protein transport from dorsal root ganglion cells tothe spinal cord are expected to be capable of suppressing peripheralneuropathy-associated pain.

In the screening method of the present invention, it is also preferredto select a substance capable of inhibiting FLRT3 binding to Unc5B.Since FLRT3 is reported to bind to the Unc5B receptor (Non PatentLiterature 7 and 9), a substance capable of inhibiting FLRT3 binding toUnc5B is expected to be capable of inhibiting the downstream signalingfrom FLRT3 and thus suppressing peripheral neuropathy-associated pain.

For the selection of the substance capable of inhibiting FLRT3expression in the screening method of the present invention, thescreening method can comprise, for example, the following steps:

bringing a test substance into contact with cells expressing FLRT3;

measuring the level of FLRT3 expression in the cells; and

comparing the level of FLRT3 expression to that in FLRT3-expressingcells not in contact with the test substance to determine whether or notthe test substance is capable of reducing the level of FLRT3 expression.

The cells expressing FLRT3 may be cells in the living body or culturedcells. The cultured cells may be primary cultured cells or a cell line.Examples of the primary cultured cells include dorsal root ganglioncells, rostral thalamic nucleus cells, hippocampal dentate granulecells, human umbilical artery endothelial cells (HUAEC), and examples ofthe cell line include Hela cells. In addition, a FLRT3-expressingtransformant with a recombinant expression vector containing a DNAencoding FLRT3 can be used.

The method for bringing the test substance into contact with the cellsis not particularly limited and may be any method that allows thecontact of the test substance with the cells. For example, in the casewhere cultured cells are used, the contact can be achieved by, forexample, addition of the test substance to culture medium. In the casewhere the test substance is brought into contact with cells in theliving body, the contact can be achieved by systemic administration suchas oral, intravenous or intraperitoneal administration, localadministration to a target organ or tissue, or the like. In addition, itis preferable to prepare a control group not in contact with the testsubstance.

In the measurement of the level of FLRT3 expression, the level of FLRT3expression may be the protein level of FLRT3 or the mRNA level of FLRT3.The measurement of the FLRT3 protein level can be achieved by extractingproteins from the cells by a known method and quantifying FLRT3 proteinby a known method for protein measurement. Examples of the known methodfor protein measurement include western blotting, EIA, ELISA, RIA and amethod using a protein assay reagent. The measurement of the FLRT3 mRNAlevel can be achieved by extracting RNAs by a known method andquantifying FLRT3 mRNA by a known method for mRNA measurement. Examplesof the known method for mRNA measurement include northern blotting,RT-PCR, quantitative RT-PCR and RNase protection assay.

When the protein or mRNA level of FLRT3 after the contact with the testsubstance is reduced as compared with that in the control group (not incontact with the test substance), the test substance can be determinedas the desired substance. The selection criterion for the reduction ofthe protein or mRNA level of FLRT3 is not particularly limited. Forexample, when the test substance reduces the protein or mRNA level ofFLRT3 to preferably 50% or less, more preferably 25% or less of that inthe cells not in contact with the test substance, the test substance canbe determined as the desired substance.

For the selection of the substance capable of inhibiting FLRT3 transportto the spinal cord in the screening method of the present invention, thescreening method can comprise, for example, the following steps:

bringing a test substance into contact with dorsal root ganglion cellsin dorsal root ganglion tissue;

measuring the level of FLRT3 in dorsal root fibers or spinal cord; and

comparing the level of FLRT3 to that in dorsal root fibers or spinalcord in the absence of contact with the test substance to determinewhether or not the test substance is capable of reducing the level ofFLRT3.

The screening method may be performed in vivo or in vitro.

In the case where the screening method is performed in vivo, it ispreferred to use neuropathic pain model animals (e.g., a partial sciaticnerve ligation model (see Examples) etc.). The contact of the testsubstance with the dorsal root ganglion cells in the dorsal rootganglion tissue can be achieved by systemic administration such as oral,intravenous or intraperitoneal administration, local administration to atarget tissue, or the like. In addition, it is preferable to prepare acontrol group not in contact with the test substance. After the contact,the dorsal root fibers or the spinal cord is harvested, proteins areextracted from the harvested tissue by a known method, and FLRT3 isquantified by a known method for protein measurement. Examples of theknown method for protein measurement include western blotting, EIA,ELISA, RIA and a method using a protein assay reagent.

In the case where the screening method is performed in vitro, forexample, dorsal root ganglion tissue is harvested from an experimentalanimal such as a rat and cultured in a commercial compartment chamberfor neuronal cells (manufactured by Tyler Research Corporation) toprepare an experimental system which allows separate treatment of dorsalroot ganglion cell bodies and dorsal root fibers (axons) with the testsubstance (References: Melli G, Keswani S C, Fischer A, Chen W, Hoke A.Spatially distinct and functionally independent mechanisms of axonaldegeneration in a model of HIV-associated sensory neuropathy. Brain.2006 May; 129(Pt 5): 1330-8. Epub 2006 Mar. 14, and Pazyra-Murphy M F,Segal R A. Preparation and maintenance of dorsal root ganglia neurons incompartmented cultures. J Vis Exp. 2008 Oct. 17; (20). pii: 951. doi:10.3791/951.). For the contact of the test substance with the dorsalroot ganglion cells, the test substance is added to culture medium onlyin the cell body compartment or in both the compartments. After that,the dorsal root fibers (axons) are harvested, proteins are extractedfrom the harvested tissue by a known method, and FLRT3 is quantified bya known method for protein measurement. Examples of the medium used forthe culture of the dorsal root ganglion tissue include Ham's F-12 mediumwith 2% B-27 Supplement (trade name, Life Technologies). The culture ofthe dorsal root ganglion tissue can be performed by a known culturemethod (for example, Kim S U, Tomonaga M, Ghetti B. Neurofibrillarydegeneration in cultured adult mouse neurons induced by maytansine. ActaNeuropathol. 1980; 52(2): 161-4, or Yong V W, Horie H, Kim S U.Comparison of six different substrata on the plating efficiency,differentiation and survival of human dorsal root ganglion neurons inculture. Dev Neurosci. 1988; 10(4): 222-30.) or a modified methodthereof.

When the level of FLRT3 in the dorsal root fibers or the spinal cordafter the contact with the test substance is reduced as compared withthat in the control group (not in contact with the test substance), thetest substance can be determined as the desired substance. The selectioncriterion for the reduction of the level of FLRT3 in the dorsal rootfibers or the spinal cord is not particularly limited. For example, whenthe test substance reduces the level of FLRT3 to preferably 50% or less,more preferably 25% or less of that in the control group, the testsubstance can be determined as the desired substance.

For the selection of the substance capable of inhibiting FLRT3 bindingto Unc5B in the screening method of the present invention, the screeningmethod can comprise, for example, the following steps:

bringing a test substance into contact with FLRT3 and Unc5B;

assessing FLRT3 binding to Unc5B; and

determining whether or not the test substance is capable of inhibitingFLRT3 binding to Unc5B.

The FLRT3 and the Unc5B used may be native or recombinant proteins. Inthe case where the FLRT3 and the Unc5B used are native proteins, thenative proteins can be obtained by a known method (for example, affinitycolumn method) from the culture supernatant or cell extract of cellsexpressing FLRT3 and/or Unc5B. In the case where the FLRT3 and the Unc5Bused are recombinant proteins, the recombinant proteins can be obtainedby a known method from the culture supernatant or cell extract of cellstransfected with a FLRT3 expression vector or a Unc5B expression vector.The recombinant FLRT3 protein can be produced by known recombinanttechniques with the use of genetic information obtainable from knowndatabases (e.g., DDBJ/GenBank/EMBL) (see Table 1). Information regardingthe nucleotide sequences of the genes encoding Unc5B proteins of variousanimals and the amino acid sequences of the Unc5B proteins can beobtained from known databases (e.g., DDBJ/GenBank/EMBL) with therespective accession numbers shown in Table 2, for example. Therecombinant Unc5B protein can be produced by known recombinanttechniques with the use of such genetic information.

TABLE 2 Nucleotide sequence Amino acid sequence Human NM_170744NP_734465 Monkey XM_001106162 XP_001106162 Mouse NM_029770 NP_084046 RatNM_022207 NP_071543 Guinea pig XM_003473767 XP_003473815

The method for bringing the test substance into contact with FLRT3 andUnc5B is not particularly limited. For example, a reaction systemcontaining FLRT3 and Unc5B is prepared, and the test substance is addedthereto. The contact time and temperature are not particularly limitedand can be selected as appropriate. In addition, it is preferable toprepare a control group not in contact with the test substance.

The method for assessing FLRT3 binding to Unc5B is not particularlylimited, and a known method for determining the level of FLRT3 bindingto Unc5B can be selected as appropriate. For example, ELISA,fluorescence polarization, etc. can preferably be used. In an exemplarymethod using ELISA, either FLRT3 or Unc5B is immobilized, the other oneand the test substance are added thereto so that the reaction proceeds,and the level of FLRT3 binding to Unc5B is determined with the use ofappropriate primary and secondary antibodies.

The method for determining whether or not the test substance is capableof inhibiting FLRT3 binding to Unc5B is not particularly limited. Whenthe level of FLRT3 binding to Unc5B after the contact with the testsubstance is reduced as compared with that in the control group (not incontact with the test substance), the test substance can be determinedas the desired substance. The selection criterion for the reduction ofthe level of FLRT3 binding to Unc5B is not particularly limited. Forexample, when the test substance reduces the level of FLRT3 binding toUnc5B to preferably 50% or less, more preferably 25% or less of that inthe absence of contact with the test substance, the test substance canbe determined as the desired substance.

Pharmaceutical Composition for Prevention or Treatment of Pain

The present invention provides a pharmaceutical composition forprevention or treatment of pain, the pharmaceutical compositioncomprising, as an active ingredient, a substance capable of inhibitingFLRT3 expression, a substance capable of inhibiting FLRT3 transport tothe spinal cord, or a substance capable of inhibiting FLRT3 binding toUnc5B. The pharmaceutical composition of the present inventionpreferably comprises, as an active ingredient, a substance selectedusing the above-described screening method of the present invention. Thepharmaceutical composition of the present invention is suitable for theprevention or treatment of peripheral neuropathy-associated pain.

The pharmaceutical composition of the present invention can be preparedin the usual manner in a dosage form containing, as an activeingredient, the substance capable of inhibiting FLRT3 expression, thesubstance capable of inhibiting FLRT3 transport to the spinal cord, orthe substance capable of inhibiting FLRT3 binding to Unc5B. For example,the dosage form can be an oral preparation and the examples includesolid or liquid preparations, specifically tablets (includingsugar-coated tablets and film-coated tablets), pills, granules, powders,capsules (including soft capsules), syrups, emulsions, suspensions, etc.These preparations can be produced by known methods and contain one ormore carriers, diluents or excipients commonly used in the field ofpharmaceutical formulation. For example, carriers or excipients used fortablets include lactose, starch, sucrose and magnesium stearate. Thedosage form may be a parenteral preparation and the examples includeinjections and suppositories. The injections include an intravenousinjection, a subcutaneous injection, an intracutaneous injection, anintramuscular injection, an intravenous infusion and an intraarticularinjection. These injections are prepared according to known methods, forexample, by dissolving, suspending or emulsifying the active ingredientin a sterile aqueous or oily liquid commonly used for injections. As anaqueous liquid for injection, for example, physiological saline, anisotonic solution containing glucose and an auxiliary substance, or thelike can be used, optionally together with a suitable solubilizer suchas alcohols (e.g., ethanol etc.), polyalcohols (e.g., propylene glycol,polyethylene glycol, etc.) and nonionic surfactants (e.g., polysorbate80, HCO-50, etc.). As an oily liquid, for example, sesame oil, soybeanoil or the like can be used, optionally together with a solubilizer suchas benzyl benzoate and benzyl alcohol. Suppositories for rectaladministration are prepared by mixing the active ingredient with acommonly used base for suppositories. The pharmaceutical preparationsthat can be obtained in the above manner are safe and less toxic, andtherefore can be administered orally or parenterally to, for example,humans and other mammals (e.g., rats, mice, rabbits, sheep, pigs, cows,cats, dogs, monkeys, etc.).

The active ingredient of the pharmaceutical composition of the presentinvention is preferably a nucleic acid capable of inhibiting FLRT3expression. Examples of the nucleic acid capable of inhibiting FLRT3expression include a siRNA (short interfering RNA), a shRNA (shorthairpin RNA) or an antisense oligonucleotide against FLRT3 gene.Information regarding the nucleotide sequence of the FLRT3 gene of ananimal to be subjected to administration of the nucleic acid can easilybe obtained from known databases (e.g., DDBJ/GenBank/EMBL). siRNA is adouble-stranded RNA of about 20 bases (for example, about 21 to 23bases) or less in length, and after expressed in cells, can inhibit theexpression of its target gene (FLRT3 gene in the present invention).shRNA is a single-stranded RNA molecule having a partial palindromicsequence, and due to complementary base pairing within the palindromicsequence, forms an intramolecular short hairpin structure of about 20base pairs or more with a 3′-terminal overhang. After introduced intocells, shRNA is intracellularly degraded to a form of about 20 bases inlength (typically for example, 21, 22 or 23 bases) and can inhibit theexpression of its target gene (FLRT3 gene in the present invention) aswith siRNA. The siRNA and the shRNA may be in any form that can inhibitFLRT3 expression. The siRNA and the shRNA can be designed based on thenucleotide sequence of the target gene by a known method. The siRNA andthe shRNA can be artificially produced by chemical synthesis.Alternatively, for example, T7 RNA polymerase and T7 promoter may beused for in vitro production of antisense or sense RNA from a templateDNA. The antisense oligonucleotide is a nucleotide that is complementaryor hybridizable to a contiguous 5 to 100 bases in the DNA sequence ofFLRT3 gene, and may be DNA or RNA. The antisense oligonucleotide may bemodified unless its function is compromised by such modification. Theantisense oligonucleotide can be produced in the usual manner, and forexample, can easily be produced with a commercial DNA synthesizer.

In the case where the active ingredient of the pharmaceuticalcomposition of the present invention is a nucleic acid capable ofinhibiting FLRT3 expression, it can be administered in the form of anon-viral or viral vector. In the case of administration in the form ofa non-viral vector, a method using liposomes to introduce nucleic acidmolecules (the liposome method, the HVJ-liposome method, the cationicliposome method, the lipofection method, the lipofectamine method,etc.), microinjection, a method using a gene gun to introduce nucleicacid molecules and a carrier (metal particles), and other methods can beused. In the case where a viral vector is used for siRNA or shRNAadministration to the living body, viral vectors such as recombinantadenoviruses and retroviruses can be used. A DNA encoding the siRNA orthe shRNA of interest is introduced into a nontoxic DNA or RNA virussuch as nontoxic strains of retrovirus, adenovirus, adeno-associatedvirus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus,Sendai virus or SV40, and the resulting recombinant virus is allowed toinfect cells or tissues for introduction of the gene of interest intothe cells or tissues.

Robinson et al. have reported that the antisense oligonucleotideconsisting of the nucleotide sequence represented by SEQ ID NO: 3(TGGGCTGATCATGGTCAGCAG) suppresses the expression of rat FLRT3(Robinson, M. Et al. FLRT3 is expressed in sensory neurons afterperipheral nerve injury and regulates neurite outgrowth. Molecular andcellular neurosciences 27, 202-14 (2004)). The target sequence of thisrat antisense oligonucleotide corresponds to the segment from positions42 to 62 of the nucleotide sequence of the human FLRT3 gene (SEQ ID NO:1), the sequence of which segment is CTGCTGACCATGATCAGCGCA (SEQ ID NO:2). Therefore, an antisense oligonucleotide targeting this segment isexpected to be useful as an active ingredient of the pharmaceuticalcomposition of the present invention.

The active ingredient of the pharmaceutical composition of the presentinvention is preferably a peptide or an antibody capable of specificallybinding to FLRT3. The binding of the antibody or the peptide to FLRT3can inhibit FLRT3 transport to the spinal cord. In addition, the bindingof the antibody or the peptide to FLRT3 can inhibit FLRT3 binding toUnc5B. The antibody capable of specifically binding to FLRT3 may be apolyclonal or monoclonal antibody. The antibody may be a whole antibodymolecule or an antibody fragment (for example, Fab, F(ab′)₂, Fab′, Fv,scFv, etc.) capable of specifically binding to an antigen of interest.The antibody is preferably a chimeric human antibody or a humanizedantibody. The antibody and the peptide can be produced by known methods.In the case where the active ingredient of the pharmaceuticalcomposition of the present invention is a peptide or an antibody, thepharmaceutical composition is preferably administered in the form of aninjection or an infusion containing the active ingredient and apharmaceutically acceptable carrier via a parenteral route, for example,intravenously, intramuscularly, intracutaneously, intraperitoneally,subcutaneously or locally.

The pharmaceutical composition of the present invention can comprise0.001 to 50% by mass, preferably 0.01 to 10% by mass, and morepreferably 0.1 to 1% by mass of the active ingredient.

The dose of the pharmaceutical composition of the present invention isappropriately determined in consideration of the purpose, the type andseverity of the disease, the age, body weight, sex and medical historyof the patient, the kind of the active ingredient, etc. In the casewhere the subject is an average human weighing about 65 to 70 kg, thedaily dose is preferably about 0.02 to 4000 mg, and more preferablyabout 0.1 to 200 mg. The total daily dose may be given as a single doseor in divided doses.

The active ingredient of the pharmaceutical composition of the presentinvention preferably acts at dorsal root ganglion cells, dorsal rootfibers or the axon terminals of dorsal root ganglion neurons, and morepreferably acts at dorsal root ganglion cells or dorsal root fibers. Inthe case where the active ingredient of the pharmaceutical compositionof the present invention is a substance capable of inhibiting FLRT3expression or a substance capable of inhibiting FLRT3 transport to thespinal cord, the active ingredient acts, for example, at dorsal rootganglia to inhibit FLRT3 expression or at dorsal root fibers to inhibitFLRT3 transport from dorsal root ganglia to the spinal cord, resultingin the prevention or treatment of pain. Therefore, the active ingredientof the pharmaceutical composition of the present invention does not haveto reach the central nervous system to exert the effects, and this is agreat advantage. In the case where the active ingredient of thepharmaceutical composition of the present invention is a substancecapable of inhibiting FLRT3 binding to Unc5B, it is preferred that theactive ingredient acts at the axon terminals of dorsal root ganglionneurons because FLRT3 is considered to bind to Unc5B at the axonterminals of dorsal root ganglion neurons in the dorsal horn of thespinal cord.

The present invention further includes the following.

-   (A) A method for prevention or treatment of pain, comprising a step    of administering an effective amount of a substance capable of    inhibiting FLRT3 expression, a substance capable of inhibiting FLRT3    transport to the spinal cord, or a substance capable of inhibiting    FLRT3 binding to Unc5B.-   (B) A substance capable of inhibiting FLRT3 expression, a substance    capable of inhibiting FLRT3 transport to the spinal cord, or a    substance capable of inhibiting FLRT3 binding to Unc5B for use in    the prevention or treatment of pain.-   (C) Use of a substance capable of inhibiting FLRT3 expression, a    substance capable of inhibiting FLRT3 transport to the spinal cord,    or a substance capable of inhibiting FLRT3 binding to Unc5B for the    production of a pharmaceutical composition for prevention or    treatment of pain.

EXAMPLES

Hereinafter, the present invention will be illustrated in detail byexamples, but the present invention is not limited thereto.

Example 1: Involvement of FLRT3 in Nociceptive Response

1-1 Experimental Methods

(1) Production of Neuropathic Pain Model

A neuropathic pain model was produced by partial sciatic nerve ligation(Seltzer Z, A novel behavioral model of neuropathic pain disordersproduced in rats by partial sciatic nerve injury. Pain 43, 205-18(1990)) and used in the experiments described later. The specificprocedure was as follows. Eight-week-old male Wistar/ST rats wereanesthetized by inhalation of a gas mixture of isoflurane and oxygen,and the left hind thigh and groin of each rat was shaved. The shavedarea was disinfected with alcohol. The skin and the muscle over thejoint connecting the thigh bone to the hip bone were incised, and thesciatic nerve, which runs along with the thigh bone, was exposed.One-third to half of the sciatic nerve was ligated with a 4-0 nylonsuture, and the muscle and skin incisions were sutured. In thecontralateral (right) hind limb, the skin and the muscle were incised,but the sciatic nerve was left intact as a sham-operated control.

(2) Measurement of Pain-Related Behavior

The von Frey filament test was used to measure the response tomechanical stimulus. Prior to testing, the pain model rats wereindividually put into a plastic cage onto a wire mesh and acclimated tothe environment for 5 to 10 minutes until settled. A filament(Semmes-Weinstein Von Frey Anesthesiometer, Muromachi Kikai Co., Ltd.)was applied to the center of the plantar surface of the hindpaw for 3 to5 seconds, and the stimulus threshold for hindpaw withdrawal (g) wasmeasured in an up-down method.

(3) Experiment of Intrathecal Administration of FLRT3 Protein

In 8-week-old male Wistar/ST rats, a polyethylene tube was inserted intothe intrathecal space from the space between the 4th and 5th lumbarvertebrae and placed with the tip of the tube in the neighborhood of thelumbar enlargement (Milligan, E. D., Hinde, J. L., Mehmert, K. K.,Maier, S. F. & Watkins, L. R. A method for increasing the viability ofthe external portion of lumbar catheters placed in the spinalsubarachnoid space of rats. Journal of neuroscience methods 90, 81-6(1999)). One week after the intubation, the von Frey filament test wasperformed to confirm whether or not the animals had intubation-causedmotor or sensory dysfunction. The animals confirmed to have nointubation-caused motor or sensory dysfunction were used in theexperiment shown below. A purified FLRT3 protein (R&D) was dissolved ata concentration of 40 ng/μL in physiological saline, and 10 μL of thissolution was infused from the outer end of the indwelling intrathecaltube. Meanwhile, 10 μL of physiological saline was administered tocontrol group animals. The von Frey filament test was performed at 12,24 and 48 hours post-administration to examine the change in withdrawalthreshold.

(4) Western Blotting

The spinal cord and the dorsal root ganglion cells of the neuropathicpain model rats were used as the samples. Each rat was anesthetized withexcess somnopentyl, and blood was completely removed by perfusion with100 mL of phosphate buffer. Lumbar vertebrae were cut, and right andleft dorsal root ganglia at the 4th to 6th lumbar levels of the spinalcord were separately harvested. The dorsal root ganglia were lysed onice with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40, 10 mMNaF, 1 mM Na₃VO₄) containing protease inhibitor cocktail (Roche). Thiscrude lysate was centrifuged at 15,000 rpm at 4° C. for 10 minutes, andthe supernatant was used as a cell lysate for the subsequent experiment.After the cell lysate was boiled in a sample buffer for 5 minutes,proteins in the cell lysate were separated by SDS-PAGE and transferredto a PVDF membrane. The membrane was blocked with 5% non-fat dry milk inPBS containing 0.05% Tween-20 (PBS-T) and incubated with an anti-FLRT3antibody (Abcam) diluted in PBS-T at 4° C. overnight. After washing withPBS-T, the membrane was incubated with an HRP-labeled anti-rabbit IgGantibody (Cell Signaling Technology). The FLRT3 protein was detectedusing an ECL chemiluminescence system (GE Healthcare). The FLRT3expression level was normalized to the beta-actin expression level.

(5) Quantitative PCR

The spinal cord and the dorsal root ganglion cells of the neuropathicpain model rats were used as the samples. Each rat was anesthetized withexcess somnopentyl, and blood was completely removed by perfusion with100 mL of phosphate buffer. Lumbar vertebrae were cut, and right andleft dorsal root ganglia at the 4th to 6th lumbar levels of the spinalcord were separately harvested. RNA was extracted with a reagent(TRIZOL, Life Technologies) from the dorsal root ganglia, and cDNA wassynthesized from the RNA with reverse transcriptase (High-capacityReverse Transcription Kit, Life Technologies). The expression level ofthe rat FLRT3 gene was quantified with TaqMan probe (Life Technologies).The expression level of the rat FLRT3 gene was normalized to theexpression level of the rat GAPDH gene (housekeeping gene).

1-2 Experimental Results

(1) FLRT3 Gene Expression Level in Neuropathic Pain Model

The FLRT3 gene expression under neuropathic pain conditions was analyzedby quantitative PCR. At 0, 4, 7, and 14 days after the production of theneuropathic pain model, spinal cord tissue (lumbar enlargement) anddorsal root ganglion cells on the side ipsilateral to the injury wereseparately harvested from each model rat. Similarly, the contralateralspinal cord tissue (lumbar enlargement) and dorsal root ganglion cellswere also harvested. RNA extracted from each harvested sample wasreverse transcribed into cDNA, and the expression of FLRT3 gene andGAPDH gene (reference) was analyzed by quantitative PCR.

The results of the quantitative PCR analysis of the dorsal root ganglioncells are shown in FIG. 1, and the results of the quantitative PCRanalysis of the spinal cord tissue (lumbar enlargement) are shown inFIG. 2. As is clear from FIG. 1, FLRT3 gene expression significantlyincreased in the dorsal root ganglion cells on the side ipsilateral tothe injury (** p<0.01 vs Day 0). In contrast, no significant increase inthe FLRT3 gene expression was observed in the spinal cord on the sideipsilateral to the injury, as is clear from FIG. 2. These results showthat, under neuropathic pain conditions, FLRT3 gene expression isincreased in the dorsal root ganglion cells, but not in the spinal cord.

(2) FLRT3 Protein Expression Level in Neuropathic Pain Model

Next, the change in FLRT3 protein expression under neuropathic painconditions was analyzed by western blotting. At 0 and 7 days after theproduction of the neuropathic pain model, spinal cord tissue (lumbarenlargement) and dorsal root ganglion cells on the side ipsilateral tothe injury were separately harvested from each model rat. Similarly, thecontralateral spinal cord tissue (lumbar enlargement) and dorsal rootganglion cells were also harvested. After the proteins from each samplewere subjected to SDS-PAGE, the expression levels of FLRT3 andbeta-actin were measured.

The results of the western blotting analysis of the dorsal root ganglioncells on day 7 post-injury are shown in FIG. 3, and the results of thewestern blotting analysis of the spinal cord tissue (lumbar enlargement)on day 7 post-injury are shown in FIG. 4. As is clear from FIG. 3, theFLRT3 protein expression in the dorsal root ganglion cells on day 7post-injury was increased on the side ipsilateral to the injury. As isclear from FIG. 4, the FLRT3 protein expression in the spinal cord onday 7 post-injury was also increased on the side ipsilateral to theinjury.

Considering all the results collectively, since the FLRT3 geneexpression level in the spinal cord was not increased by nerve injury asshown in the above-described quantitative PCR analysis, the increase inFLRT3 protein expression in the spinal cord after the injury is probablydue to the axonal transport of FLRT3 expressed in sites other than thespinal cord, in particular dorsal root ganglion cells, to the spinalcord.

(3) Potentiation of Nociceptive Response by Intrathecal Administrationof FLRT3 Protein

In order to clarify the function of FLRT3 protein in the spinal cord,the change in the nociceptive response after intrathecal administrationof FLRT3 protein was examined. The von Frey filament test was performedat 12, 24 and 48 hours post-administration to examine the change inwithdrawal threshold. The results are shown in FIG. 5. As is clear fromFIG. 5, the FLRT3 protein-administered animals showed a gradualreduction in withdrawal threshold after the administration. The degreeof the reduction in withdrawal threshold in the FLRT3protein-administered rats was significantly larger than that in thecontrol rats at 12, 24 and 48 hours post-administration (Tukey-Kramertest, ** p<0.01, * p<0.05).

The above results indicate that FLRT3 functions to potentiatenociceptive response in the spinal cord of adult animals, thus causingpain.

Example 2: Suppression of Nociceptive Response by Anti-FLRT3 Antibody

In order to clarify whether anti-FLRT3 antibody has analgesic effect onneuropathic pain, an anti-FLRT3 antibody (R&D, AF2795) was intrathecallyadministered to model rats which had developed pain, and pain-relatedbehavior in the rats was examined. First, a polyethylene tube wasinserted into the intrathecal space in 8-week-old male Wistar rats. Oneweek after the intubation, the sciatic nerve of the left hind limb ofeach rat was partially ligated to produce a neuropathic pain model.Seven days after the pain model production, the von Frey filament testwas performed to confirm the reduction in withdrawal threshold (i.e.,the development of mechanical allodynia) in each rat. The anti-FLRT3antibody was dissolved at a concentration of 1 μg/μL in physiologicalsaline, and 30 μL of this solution was infused from the outer end of theindwelling intrathecal polyethylene tube. Meanwhile, the same volume ofa control goat IgG solution (1 μg/μL) was administered to control groupanimals. After the administration, the outer end of the tube was closedand the skin incision was sutured. The von Frey filament test wasperformed at 12, 24 and 48 hours post-administration to examine thechange in withdrawal threshold in the hind limb ipsilateral to theinjury.

The results are shown in FIG. 6. As shown in FIG. 6, the withdrawalthreshold in the anti-FLRT3 antibody-administered group was higher thanthat in the control group. In particular, significant difference wasobserved at 24 hours post-administration as compared with the controlgroup (Tukey-Kramer test, * p<0.05). These results show that theintrathecal administration of the anti-FLRT3 antibody exerts analgesiceffect on neuropathic pain.

The present invention is not limited to the particular embodiments andexamples described above, and various modifications can be made withinthe scope of the appended claims. Other embodiments provided by suitablycombining technical means disclosed in separate embodiments of thepresent invention are also within the technical scope of the presentinvention. All the academic publications and patent literature cited inthe description are incorporated herein by reference.

The invention claimed is:
 1. A screening method for a pain suppressorsubstance, the method comprising: (a) contacting cells that expressFLRT3 with a test substance that reduces the level of FLRT3 expression,as compared to a control population of FLRT3-expres sing cells that werenot contacted with said test substance; (b) measuring a reduction ofFLRT3 expression in the cells that express FLRT3 after (a), as comparedto a control population of FLRT3-expressing cells that were notcontacted with said test substance; and (c) incorporating said testsubstance, which reduces the FLRT3 expression measured in (b), into apain suppressor pharmaceutical composition.
 2. The method according toclaim 1, wherein the pain suppressor pharmaceutical compositionsuppresses peripheral neuropathy-associated pain.
 3. The methodaccording to claim 1, wherein the test substance is a pain repressorsubstance that inhibits FLRT3 transport to the spinal cord.
 4. Themethod according to claim 1, wherein the test substance is a painrepressor substance that inhibits FLRT3 binding to Unc5B.
 5. The methodaccording to claim 1, wherein: the cells that express FLRT3 and thecontrol population of FLRT3-expressing cells are dorsal root ganglioncells of the dorsal root ganglion tissue or spinal cord; and the levelof FLRT3 expression in the dorsal root ganglion tissue or spinal cord ismeasured, as compared to the control population of FLRT3-expressingcells that were not contacted with said test substance.
 6. The method ofclaim 1, wherein the pain suppressor pharmaceutical composition is anoral preparation, a parenteral preparation, a suppository or isincorporated into a liposome.
 7. The method of claim 1, wherein the testsubstance is a nucleic acid, which inhibits FLRT3 expression.
 8. Themethod of claim 7, wherein the nucleic acid, which inhibits FLRT3expression, is a short interfering RNA (siRNA), a short hairpin RNA(shRNA), or an antisense oligonucleotide specific for the FLRT3 gene. 9.The method of claim 1, wherein the pain suppressor pharmaceuticalcomposition comprises 0.01% to 10% by mass of the test substance.